Posted by Li-Huei Tsai, director of Picower Institute for Learning and Memory (Massachusetts Institute of Technology) and her coworkers recently filed a patent application (US2022288104) to reverse apolipoprotein E gene (called APOE4) associated cholesterol phenotype by administering cyclodextrin (HPBCD) to inhibit amyloid synthesis (blog entry author note: the basis of the amyloid theory was recently challenged in Science, doi: 10.1126/science.ade0209.)
About 25% of people carry one copy of APOE4, and 2 to 3% carry two copies. Though APOE4 is the strongest risk factor gene for Alzheimer’s disease, although inheriting APOE4 does not mean a person will definitely develop the disease.
APOE4 expression in oligodendrocytes causes both cholesterol accumulation and impaired myelination. It was reasoned by Prof. Tsai that inhibiting cholesterol biosynthesis and/or facilitating cholesterol transport in APOE4 oligodendrocytes may reduce intracellular accumulation and improve myelination. To test this hypothesis, small molecules that inhibit cholesterol biosynthesis (simvastatin and atorvastatin) and 2-hydroxypropyl-β-cyclodextrin, a cholesterol-solubilizing agent known to facilitate cholesterol transport and reduce intracellular cholesterol accumulation associated with Niemann-Pick disease type C, were employed. It was first assessed whether simvastatin, atorvastatin, or HPBCD could reduce intracellular cholesterol by treating APOE4 iPSC-derived oligodendroglia with each drug for two weeks and subsequently quantifying the total amount of bodipy-cholesterol staining per condition. APOE4 oligodendroglia cultured in the presence of either cholesterol biosynthesis inhibitors (either simvastatin or atorvastatin) exhibited elevated bodipy-cholesterol staining that was significantly (p=0.0085 and 0.0450) higher than APOE3/3 oligodendroglia. However, APOE4 oligodendroglia treated with HPBCD exhibited reduced bodipy-cholesterol staining that was not significantly different (p>0.9999) from that in APOE3 oligodendroglia. In HPBCD-treated APOE4 oligodendroglia, a significant (p=0.0092) reduction of the number of intracellular droplets of bodipy-cholesterol was observed as well as a significant (p<0.0001) reduction of neutral lipid droplets measured by bodipy staining, suggesting that HPBCD reduced both intracellular cholesterol and neutral lipid accumulation such as triacylglycerides. HPBCD treatment also led to significant (p=0.0011) upregulation of expression CYP46A1, the primary enzyme that hydroxylates cholesterol in the brain, which facilitates cholesterol trafficking and clearance. In APOE4 oligodendroglia treated with atorvastatin or simvastatin expression of CYP46A1 was not significantly different (p=0.4324; 0.0571) from untreated APOE4 controls.
It was also assessed whether HPBCD could reduce aberrant cholesterol accumulation in oligodendrocytes and promote increased myelination in APOE4KI mice in vivo. APOE4KI mice were treated with subcutaneous injections of HPBCD or saline (control) for eight weeks (n=5 control and 4 HPBCD treated mice). In the post-mortem human brain from APOE4-carriers, increased cholesterol accumulations surrounding Olig2-positive nuclei was observed. In the hippocampus of APOE4KI mice treated with HPBCD, bodipy-cholesterol staining that accumulated around Olig2-positive nuclei was significantly (p=0.0038) reduced compared to control mice. Moreover, in HPBCD-treated mice, a significant (p=0.0423) increase in bodipy-cholesterol staining co-localizing within 1 μm of Mbp staining was observed, indicating that HPBCD facilitates increased trafficking of cholesterol to the myelin sheath. Encouragingly APOE4KI mice treated with HPBCD-treated mice exhibited significantly (p=0.0008) increased Mbp staining, suggesting that HPBCD may increase myelin levels in APOE4 mice. To more directly assess myelination in HPBCD treated APOE4 mice, TEM-based ultrastructural analysis was performed. APOE4 mice treated with HPBCD had a significantly (p<0.0001) lower g-ratio (n=150 neurons for each genotype) than control mice indicating an increased number of myelinated axons and thicker myelin sheaths in HPBCD treated mice. Collectively, this data demonstrates that pharmacologically enhancing cholesterol transport in APOE4-carriers can increase myelination.
To investigate whether the increased myelination observed in HPBCD treated APOE4KI mice could promote functional improvements, the novel object recognition assay was performed to evaluate learning and memory. During an open field task, control (n=12) and HPBCD-treated (n=14) APOE4KI mice exhibited similar total distance moved (p=0.1275), velocity (p=0.1711), and duration in the center of the cages (p=0.4478), suggesting HPBCD treatment did not affect locomotor or anxiety phenotypes. However, when exposed to a novel object, APOE4KI mice treated with HPBCD had a significantly (p=0.0466) increased preference for interacting with the novel object than control APOE4KI mice, suggesting that cyclodextrin treatment may improve learning and memory.
These findings are in line with previous observations that HPBCD may act as an active agent and may ameliorate neurological disorders. It is remarkable in the work of Professor Tsai that such effect was demonsrated with cyclodextrin applied subcutaneously instead of direct injection to cerebrospinal fluid. Consequently, addressing the question whether HPBCD can pass through the blood brain barrier is more actual than ever.
One comment